(1) State of the Invention
This invention relates to a method of generating light (chemiluminescence) by the action of a peroxidase enzyme with an oxidant such as hydrogen peroxide on a group of hydroxyaryl cyclic diacylhydrazides. The invention also relates to a method of greatly increasing the amount of chemiluminescence produced from this process by the use of specific enhancers. The invention also relates to the use of this method to detect the peroxidase enzyme. The invention also relates to the use of this method to detect hydrogen peroxide. Further, the invention relates to the use of the method to detect and quantitate various biological molecules. For example, the method may be used to detect haptens, antigens and antibodies by the technique of immunoassay, proteins by Western blotting, DNA and RNA by Southern and Northern blotting, respectively. The method may also be used to detect DNA in DNA sequencing applications. The method may additionally be used to detect enzymes which generate hydrogen peroxide such as glucose oxidase, glucose-6-phosphate dehydrogenase, galactose oxidase, galactose-6-phosphate dehydrogenase, and amino acid oxidase.
(2) Prior Art
a. Chemiluminescent Oxidation of Hydroxyaryl Cyclic Diacylhydrazides. A series of publications have appeared concerning the chemiluminescent reactions of the hydroxyphthalhydrazides, 5- and 6-hydroxy-2,3-dihydrophthalazine-1,4-dione with different chemical oxidizing agents (I. E. Kalinichenko, A. T. Pilipenko, V. A. Barovskii, Ukr. Khim. Zh. (Russ. Ed.), 43(10), 1102-6 (1977); Kalinichenko, I. E.; Barovskii, V. A.; Pilipenko, A. T., Ukr. Khim. Zh. (Russ. Ed.), 44(7), 748-52 (1978); A. T. Pilipenko, V. A. Barovskii, I. E. Kalinichenko, Zh. Anal. Khim., 33(10), 1880-4 (1978); I. E. Kalinichenko, V. A. Barovskii, Ukr. Khim. Zh. (Russ. Ed.), 45(1), 58-62 (1979); I. E. Kalinichenko, T. M. Tkachuk, A. T. Pilipenko, Zh. Anal. Khim., 39(7), 1281-4 (1984)). Gundermann reports the chemiluminescent oxidation of 7-hydroxynaphthalene-1,2-dicarboxylic hydrazide in aqueous solution with hydrogen peroxide and a metal catalyst (K.-D. Gundermann, W. Horstmann, G. Bergman, Lieb. Ann. der Chem., 684, 127-141 (1965)). No publications are known concerning the use of hydroxyaryl cyclic diacylhydrazides with a peroxidase enzyme to generate chemiluminescence. Further, there are no known reports of the use of hydroxyaryl cyclic diacylhydrazides to generate chemiluminescence for the detection of biological compounds.
b. Chemiluminescent Oxidation of Luminol and Related Compounds. Aminoaryl cyclic diacylhydrazides such as luminol and isoluminol react with hydrogen peroxide and a peroxidase enzyme catalyst (such as horseradish peroxidase, HRP) under basic conditions with emission of light. The reaction is also catalyzed by small amounts of several metal ions including Fe(III), Cu(II) and Cr(III) or iron-containing organic compounds (e.g. R. B. Brundett D. F. Roswell, E. H. White, J. Am. Chem. Soc., 94, 7536 (1972)). This reaction has been used as the basis for analytical methods for the detection of hydrogen peroxide and for metal ions. Luminol and isoluminol may be directly conjugated to a species to be detected. The first chemiluminescent immunoassay using luminol as a label was reported by Schroeder for an assay of biotin. (H. R. Schroeder, P. O. Vogelhut, R. J. Carrico, R. C. Boguslaski, R. T. Buckler, Anal. Chem. 48, 1933 (1976). Several applications of the use of luminol derivatives as labels have been reported since then (H. R. Schroeder in Luminescent Immunoassays: Perspectives in Endocrinology and Clinical Chemistry, M. Serio and M. Pazzagli, Eds., Raven Press, New York, pp 129-146 (1982); M. Pazzagli, G. Messeri, A. L. Caldini, G. Monetti, G. Martinazzo, M. Serio, J. Steroid Biochem., 19, 407 (1983); J. De Boever, F. Kohen and D. Vandekerckhove in Bioluminescence and Chemiluminescence New Perspectives, J. Scholmerich, et al, Eds., J. Wiley & Sons, Chichester, pp 257-260 (1987)). Various enhancers have also been employed in conjunction with the use of luminol to increase the intensity of light emitted. These include D-luciferin (T. P. Whitehead, G. H. Thorpe, T. J. Carter, C. Groucutt, L. J. Kricka, Nature, 305, 158 (1983)) and p-iodophenol and p-phenylphenol (G. H. Thorpe, L. J. Kricka, S. B. Mosely, T. P. Whitehead, Clin. Chem., 31, 1335 (1985)). U.S. Pat. No. 4,834,918 to Wulff et al describes the use of fluorescein as an enhancer.
c. Enzyme-Catalyzed Chemiluminescent Reactions
(1) Enzymatic Triggering of Stabilized 1,2-Dioxetanes. Recently developed thermally stable dioxetanes can be triggered by chemical and enzymatic processes to generate chemiluminescence on demand (A. P. Schaap, U.S. Pat. No. 4,857,652; A. P. Schaap, R. S. Handley, B. P. Giri, Tetrahedron Lett., 935 (1987); A. P. Schaap, T. S. Chen, R. S. Handley, R. DeSilva, B. P. Giri, Tetrahedron Lett., 1155 (1987); and A. P. Schaap, M. D. Sandison, R. S. Handley, Tetrahedron Lett., 1159 (1987); Bronstein, B. Edwards, J. C. Voyta, J. Biolumin. Chemilumin. 4, 99-111 (1989); A. P. Schaap, H. Akhavan, L. J. Romano, Clin. Chem., 35, 1863 (1989); D. Pollard-Knight, A. C. Simmonds, A. P. Schaap, H. Akhavan, M. A. W. Bradley, Anal. Biochem. 185,353-358 (1990); J. M. Clyne, J. A. Running, R. Sanchez-Pescador, D. Besemer, M. Stempien, A. P. Schaap, R. S. Stephens, M. S. Urdea, J. Biolumin. Chemilumin. 2, 193 (1988)).
(2) Enzymatic Generation of Hydrogen Peroxide. Various enzymatic reaction schemes are known which produce hydrogen peroxide. The generated hydrogen peroxide can, in turn, be used to oxidize a compound which emits light. For example, glucose oxidase reacts with O.sub.2 and sucrose to produce hydrogen peroxide. Similarly, amino acid oxidase reacts with an amino acid and O.sub.2 to produce hydrogen peroxide. Examples of compounds which are oxidized by hydrogen peroxide to produce light are luminol and isoluminol, lucigenin, esters of N-methylacridine and esters and amides of oxalic acid. Glucose-6-phosphate dehydrogenase and galactose-6-phosphate dehydrogenase have been used to produce H.sub.2 O.sub.2 indirectly by reduction of oxygen through an electron-relay system. (K. Tanabe, T. Kawasaki, M. Maeda, A. Tsuji, M. Yabuuchi, Bunseki Kagaku, 36, 82 (1987), A. Tsuji, M. Maeda, H. Arakawa, Anal. Sci., 5, 497 (1989)).
(3) Enzymatic Generation of Luminol from a Luminol-NAG Conjugate. The compound o-aminophthalhydrazide-N-acetyl-.beta.-D-glucosaminide (luminol-NAG) and 4'-(6'-diethylaminobenzofuranyl)-phthalhydrazide-N-acetyl-.beta.-D-glucosa minide are substrates for the enzyme N-acetyl-.beta.-D-glucosaminidase which serve as a masked form of luminol. Upon action of the enzyme on these substrates, luminol or a luminol derivative are liberated which may be detected as described above. (K. Sasamoto, Y. Ohkura, Chem. Pharm. Bull. 38(5), 1323 (1990); K. Sasamoto, Y. Ohkura, Chem. Pharm. Bull. 39(2), 411 (1991)).
(4) Bioluminescent Detection of Firefly or Bacterial Luciferase. A class of enzymes known as luciferases, catalyze the autoxidation of certain substrates known as luciferins in a number of living organisms. An example is the firefly whose bioluminescent process oxidizes its luciferin to produce light with 88% efficiency. A DNA dot-hybridization assay has been reported in which alkaline phosphatase was employed to generate free firefly luciferin from the phosphate salt. Luciferase added in the final step caused the chemiluminescent oxidation of the luciferin (R. Hauber, R. Geiger, Nuc. Ac. Res., 16(32), 1213 (1988)).
d. Use of Chemiluminescence in Enzyme Immunoassays and DNA Hybridization assays. Biological assays such as enzyme immunoassays and DNA probe assays involving enzymes utilize a wide variety of substrates which either form a color (chromogenic), become fluorescent (fluorogenic) or emit light (chemiluminogenic) upon reaction with the enzyme. Of these three choices, chemiluminescence offers the greatest sensitivity. In an assay, the enzyme (reporter enzyme) is conjugated or bound to the molecule to be detected or to some other substance capable of selectively binding or associating with the molecule to be detected. Once the bound reporter enzyme is separated from unbound enzyme, a substrate is provided with which the reporter enzyme generates a signal. Chemiluminogenic substrates used to date include enzyme-triggerable dioxetanes such as the alkaline phosphatase substrate LUMIGEN PPD (A. P. Schaap, H. Akhavan, L. Y. Romano, Clin. Chem., 35, 1863 (1989)). This substrate has been used extensively in enzyme-linked immunoassays and DNA probes. The enzyme horseradish peroxidase has been widely used in enzyme immunoassays and DNA hybridization assays with chemiluminescent detection using luminol or isoluminol as substrate (T. P. Whitehead, G. H. Thorpe, T. J. Carter, C. Groucutt, L. J. Kricka, Nature, 305, 158 (1983), G. H. Thorpe, L. J. Kricka, S. B. Mosely, T. P. Whitehead Clin. Chem., 31, 1335 (1985), G. H. Thorpe, S. B. Mosely, L. J. Kricka, R. A. Stott, T. P. Whitehead Anal. Chim. Acta, 170, 107 (1985), J. A. Matthews, A. Batki, C. Hynds, L. J. Kricka, Anal. Biochem., 151, 205, (1985)). A general discussion appears in Chemiluminescence, A. K. Campbell, Ellis Harwood, Chichester, England 80 and 441, 446-450, 457 (1988). Commercially available kits for conjugation of HRP with enhanced luminol chemiluminescent detection are sold under the tradename AMERLITE.